Glucosyl stevia composition

ABSTRACT

Glucosyl stevia compositions are prepared from steviol glycosides of  Stevia rebaudiana  Bertoni. The glucosylation was performed by cyclodextrin glucanotransferase using starch as the source of glucose residues. The short-chain glucosyl stevia compositions were purified to &gt;95% content of total steviol glycosides. The compositions can be used as sweetness enhancers, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of and claims thebenefit of priority from U.S. patent application Ser. No. 13/029,263,filed on Feb. 17, 2011 and U.S. patent application Ser. No. 13/074,179,filed on Mar. 29, 2011.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a process for producing a highly purified foodingredient from the extract of the Stevia rebaudiana Bertoni plant andits use in various food products and beverages.

2. Description of the Related Art

Nowadays sugar alternatives are receiving increasing attention due toawareness of many diseases in conjunction with consumption of high-sugarfoods and beverages. However many artificial sweeteners such as dulcin,sodium cyclamate and saccharin were banned or restricted in somecountries due to concerns on their safety. Therefore non-caloricsweeteners of natural origin are becoming increasingly popular. Thesweet herb Stevia rebaudiana Bertoni, produces a number of diterpeneglycosides which feature high intensity sweetness and sensory propertiessuperior to those of many other high potency sweeteners.

The above-mentioned sweet glycosides, have a common aglycon, steviol,and differ by the number and type of carbohydrate residues at the C13and C19 positions. The leaves of Stevia are able to accumulate up to10-20% (on dry weight basis) steviol glycosides. The major glycosidesfound in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%),and Rebaudioside C (1-2%). Other glycosides such as Rebaudioside B, D,E, and F, Steviolbioside and Rubusoside are found at much lower levels(approx. 0-0.2%).

Two major glycosides—Stevioside and Rebaudioside A, were extensivelystudied and characterized in terms of their suitability as commercialhigh intensity sweeteners. Stability studies in carbonated beveragesconfirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983)Stability studies of stevioside and Rebaudioside A in carbonatedbeverages. J. Agric. Food Chem. 31: 409-412.)

Steviol glycosides differ from each other not only by molecularstructure, but also by their taste properties. Usually stevioside isfound to be 110-270 times sweeter than sucrose, Rebaudioside A between150 and 320 times, and Rebaudioside C between 40-60 times sweeter thansucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside Ahas the least astringent, the least bitter, and the least persistentaftertaste thus possessing the most favorable sensory attributes inmajor steviol glycosides (Tanaka O. (1987) Improvement of taste ofnatural sweetners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989)Stevia: steps in developing a new sweetener. In: Grenby T. H. ed.Developments in sweeteners, vol. 3. Elsevier Applied Science, London.1-43.)

Methods for the extraction and purification of sweet glycosides from theStevia rebaudiana plant using water or organic solvents are describedin, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938;5,972,120; 5,962,678; 7,838,044 and 7,862,845.

However, even in a highly purified state, steviol glycosides stillpossess undesirable taste attributes such as bitterness, sweetaftertaste, licorice flavor, etc. One of the main obstacles for thesuccessful commercialization of stevia sweeteners are these undesirabletaste attributes. It was shown that these flavor notes become moreprominent as the concentration of steviol glycosides increases (PrakashI., DuBois G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008)Development of rebiana, a natural, non-caloric sweetener. Food Chem.Toxicol., 46, S75-S82.)

Some of these undesirable properties can be reduced or eliminated bysubjecting steviol glycosides to the reaction of intermoleculartransglycosylation, when new carbohydrate residues are attached toinitial molecule at C13 and C19 positions. Depending on the number ofcarbohydrate residues in these positions the quality and potency of thecompounds taste will vary.

Pullulanase, isomaltase (Lobov S. V., Jasai R., Ohtani K., Tanaka O.Yamasaki K. (1991) Enzymatic production of sweet stevioside derivatives:transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965),β-galactosidase (Kitahata S., Ishikawa S., Miyata T., Tanaka O. (1989)Production of rubusoside derivatives by transglycosylation of variousβ-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextransaccharase (Yamamoto K., Yoshikawa K., Okada S. (1994) Effectiveproduction of glucosyl-stevioside by α-1,6-transglucosylation of dextrandextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used astransglycosylating enzymes, together with pullulan, maltose, lactose,and partially hydrolyzed starch, respectively, as donors of glycosidicresidues.

The transglucosylation of steviol glycosides was also performed byaction of cyclodextrin glucanotransferases (CGTase) produced by Bacillusstearotherinophilus (U.S. Pat. Nos. 4,219,571, and 7,807,206) as aresult α-1,4-glucosyl derivatives were formed with degree ofpolymerization up to 10.

It was shown (Tanaka O. (1987) Improvement of taste of naturalsweetness. Pure Appl. Chem. 69:675-683) that the taste profile andsweetness power of glucosyl derivatives are largely dependent on numberof additional α-1,4-glucosyl derivatives, i.e. the degree ofpolymerization of the α-1,4-glucosyl chain. However, in most oftransgucosylated stevia products the degree of polymerization is usuallybelow nine. As with any reaction, the transglucosylation reaction isinhibited by its products, and further increase of α-1,4-glucosylresidues is inhibited by reaction products, particularly short-chainmaltooligosaccharides.

Therefore it is necessary to develop simple process of preparation ofhigh purity glucosyl stevia products with greater α-1,4-glucosyl chainlength and better taste profile.

SUMMARY OF THE INVENTION

The present invention is aimed to overcome the disadvantages of existingStevia sweeteners. The invention describes a process for producing ahigh purity food ingredient from the extract of the Stevia rebaudidanaBertoni plant use thereof in various food products and beverages as asweetness and flavor modifier.

The invention, in part, pertains to an ingredient comprisingglucosylated derivatives of steviol glycosides of Stevia rebaudianaBertoni plant. The steviol glycodsides are selected from the groupconsisting of stevioside, Rebaudioside A, Rebaudioside B, RebaudiosideC, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A,steviolbioside, rubusoside, as well as other steviol glycosides found inStevia rebaudiana Bertoni plant and mixtures thereof.

The invention, in part, pertains to a process for producing aningredient containing glucosylated forms of stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, as well asother steviol glycosides found in Stevia rebaudiana Bertoni plant. Theprocess can be an enzymatic transglucosylating process using CGTasesproduced by cultures of Bacillus stearothermophilus. The process mayinclude the steps of decolorizing, desalting and removingmaltooligosaccharides. The decolorizing can be performed using activatedcarbon. The desalting can be performed by passing through ion exchangeresins and/or membrane filters. Removing the maltooligosaccharides canbe performed by passing through macroporuos polymeric resin.

In the invention, Stevia extract commercialized by PureCircle (JiangXi)Co., Ltd. (China), containing stevioside (28-30%), Rebaudioside A(50-55%), Rebaudioside C (9-12%), Rebaudioside F (1-3%) and otherglycosides amounting to total steviol glycosides' content of at least95%, was used as a starting material. Alternatively stevia extracts withdifferent ratio of steviol glycosides as well as highly purified steviolglycosides such as Rebaudioside A, stevioside, Rebaudioside D,rubusoside etc, may be used as starting materials.

The starting material was subjected to enzymatic transglucosylation byaction of cyclodextrin glycosyltransferase (CGTase) in the presence ofstarch as a glucose donor. As a result α-1,4-glucosyl derivatives wereformed with a degree of polymerization up to 9. Then themaltooligosaccharides from obtained reaction mixture were removed byAmberlite XAD7 HP resin. The resulting mixture of α-1,4-glucosylderivatives (with a degree of polymerization up to 9) was subjected tothe second enzymatic transglucosylation by CGTase in the presence ofstarch as a glucose donor. As a result of the second glucosylation,α-1,4-glucosyl derivatives with a degree of polymerization up to 20 wereformed. The maltooligosaccharides obtained during second glucosylationwere removed by Amberlite XAD7 HP resin. Then the obtained mixture ofα-1,4-glucosyl derivatives (with degree of polymerization up to 20) wasdecolorized, deionized, concentrated and spray dried.

The obtained products were applied in various foods and beverages assweeteners, sweetener enhancers and flavor modifiers, including icecream, cookies, bread, fruit juices, milk products, baked goods andconfectionary products.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows a high-performance liquid chromatogram of transglucosylatedStevia extract containing α-1,4-glucosyl-derivatives with up to nineα-1,4-glucosyl residues;

FIG. 2 shows a high-performance liquid chromatogram of transglucosylatedStevia extract containing α-1,4-glucosyl-derivatives with up to twentyα-1,4-glucosyl residues.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Stevia extract commercialized by PureCircle (JiangXi) Co., Ltd. (China),containing stevioside (28-30%), Rebaudioside A (50-55%), Rebaudioside C(9-12%), Rebaudioside F (1-3%) and other glycosides (hereinaftercollectively, “steviol glycosides”) amounting to total steviolglycosides content of at least 95%, was used as a starting material.Alternatively stevia extracts with different ratio of steviol glycosidesas well as highly purified steviol glycosides such as Rebaudioside A,stevioside, Rebaudioside D, rubusoside etc, may be used as startingmaterials.

The HPLC analysis of the raw materials and products was performed onAgilent Technologies 1200 Series (USA) liquid chromarograph, equippedwith Zorbax-NH₂ (4.6×250 mm) column. The mobile phase wasacetonitrile-water gradient from 80:20, v/v (0-2 min) to 50:50, v/v(2-70 min). A diode array detector set at 210 nm was used as thedetector.

The transglucosylation was accomplished by cyclomaltodextringlucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillusstearothermophilus St-100 (PureCircle Sdn Bhd Collection of IndustrialMicroorganisms—Malaysia). However, any other CGTase or enzyme possessingintermolecular transglucosylation activity may be applied as well. Theenzyme can be in a form of cell-free culture broth, concentrated liquidcell-free culture broth, spray dried or freeze dried cell-free culturebroth, or high purity protein. Free and immobilized enzyme preparationscan be used.

The activity of CGTase preparations was determined according to theprocedure described in Hale W. S., Rawlins L. C. (1951) Amylase ofBacillus macerans. Cereal Chem. 28, 49-58.

Starches of different origin may be used as donors of glucosyl unitssuch as, derived from wheat, corn, potato, tapioca, and sago.

Starch was subjected to partial hydrolysis (liquefaction) prior to thetransglycosylation reaction. The dextrose equivalent of the partiallyhydrolyzed starch can be in the range of about 10-25, preferably about12-16. Any enzyme capable of starch hydrolysis may be used forliquefaction, such as α-amylases, β-amylases etc. In one embodiment,CGTase and α-amylase mixtures as liquefying enzymes are preferred.

α-Amylase activity is expressed in Kilo Novo α-amylase Units (KNU). OneKNU is the amount of α-amylase which, under standard conditions (pH 7.1;37° C.), dextrinizes 5.26 g starch dry substance per hour.

The liquefaction mixture contains about 0.001-0.2 KNU, preferably about0.05-0.1 KNU of α-amylase per one unit of CGTase.

The use of α-amylase in liquefaction allows achieving higher throughputsin further activated carbon filtration. When the CGTase is used as theonly liquefying enzyme the filtration rate is approximately 10-15 L/hrper 1 m² of filter surface. In case of liquefaction enzyme mixture(comprising α-amylase and CGTase) the filtration rate is twice asfast—approximately 20-30 L/hr per 1 m² of filter surface.

The ratio of starch and CGTase in the liquefaction mixture is about0.1-0.5 units per one gram of starch, preferably about 0.2-0.4 units pergram.

The concentration of starch in liquefaction mixture is about 15-40%(wt/wt), preferably about 20-30%.

The liquefaction is conducted at about 70-90° C. during about 0.5-5hours, preferably about 1-2 hours.

After liquefaction, the reaction mixture is subjected to thermalinactivation of α-amylase at low pH conditions. The preferred pH rangefor inactivation is about pH 2.5 to pH 3.0 and preferred temperature isabout 95-105° C. The duration of thermal inactivation is about 5-10minutes.

After the inactivation, the pH of the reaction mixture is adjusted toabout pH 5.5-6.5 and the steviol glycosides are added to the mixture anddissolved. The preferred ratio of steviol glycosides to starch (kg ofsteviol glycosides per 1 kg of starch) is about 0.5-1.5, preferablyabout 0.8-1.2.

A second portion of CGTase preparation is added and the firsttransglucosylation reaction is conducted at about 65° C. for about 24-48hours. The amount of the second portion of CGTase is about 0.2-4 unitsof CGTase per gram of solids, preferably about 0.5-1.2 units per gram ofsolids.

Upon completion of transglucosylation the reaction was stopped byheating at about 95° C. for about 15 minutes to inactivate the enzyme.As a result a mixture of α-1,4-glucosyl derivatives with a degree ofpolymerization up to 9 was obtained. In order to remove the short chainmaltoologisaccharides, which inhibit the further elongation ofα-1,4-glucosydic chain, the reaction mixture was passed through a columnpacked with Amberlite XAD7 HP macroporous adsorbent resin. The steviolglycosides and their glucosylated derivatives were adsorbed on the resinand subsequently eluted by aqueous ethanol. The resulting aqueousethanol eluate, containing glucosyl steviol glycosides, was evaporated,concentrated and spray dried to obtain transglucosylated Stevia extractcontaining glucosyl derivatives with up to nine α-1,4-glucosyl residues.

A second portion of starch was subjected to partial hydrolysis(liquefaction) as described above.

After the liquefaction the transglucosylated Stevia extract obtainedduring the first glucosylation (with up to nine α-1,4-glucosyl residues)was added. The preferred ratio of transglucosylated Stevia extract tostarch (kg of transglucosylated Stevia extract per 1 kg of starch) isabout 0.5-1.5, preferably about 0.8-1.2.

Another portion of the CGTase preparation was added and the secondtransglucosylation reaction was conducted at about 65° C. for about24-48 hours. The amount of this portion of CGTase is about 0.2-4 unitsof CGTase per gram of solids, preferably about 0.5-1.2 units per gram ofsolids.

Upon completion of transglucosylation the reaction was stopped byheating at about 95° C. for about 15 minutes to inactivate the enzyme.As a result a mixture of α-1,4-glucosyl derivatives with a degree ofpolymerization up to 20 was obtained.

The maltooligosaccharides were removed from reaction mixture usingAmberlite XAD7 HP resin as described above. The steviol glycosides andtheir glucosylated derivatives were adsorbed on the resin andsubsequently eluted by aqueous ethanol. The resulted aqueous ethanoleluate, containing glucosyl steviol glycosides, was treated withactivated carbon, to obtain decolorized reaction mixture. The amount ofactivated carbon was about 0.02-0.4 grams per gram of solids, preferablyabout 0.05-0.2 grams per gram of solids. The decolorized solution wasfurther desalted by passing through ion exchange resins, such asAmberlite FPC23 (H⁺ type) and Amberlite FPA51 (OH⁻ type). Otherappropriate decolorizing and desalting methods, such as membranefiltration, or other methods known in the art can be used.

The desalted reaction mixture was further concentrated by vacuumevaporator and dried by means of a spray dryer. Other appropriateconcentrating and drying methods, such as membrane filtration, freezedrying, or other methods known to art can be used.

The resulting product was transglucosylated Stevia extract containingα-1,4-glucosyl-derivatives with up to twenty α-1,4-glucosyl residues(Sample 2).

The transglucosylated Stevia extract can optionally be further purifiedby removing unreacted steviol glycosides. The dried transglucosylatedStevia extract powder is suspended in aqueous alcohol. The powder toaqueous alcohol ratio (wt/vol) can range from 1:1 to 1:20, preferably1:3 to 1:10. The aqueous alcohol contains 0-50% (vol), preferably 1-10%water. The suspension is agitated at 30-100° C., preferably 50-85° C.during 1-24 hours, preferably 2-15 hours. Then the suspended solids areseparated by means of filtration. Any other technique known in the artsuitable for separating suspended solids from liquid such ascentrifugation, decanting, etc. can be used. The obtained solids aredried in rotary drum vacuum drier. Any other dryer known t in the artmay be used as well. Alternatively the separated solids may be dissolvedin water, evaporated from traces of alcohol and spray dried.

The alcohols employed in this optional step may be selected from thegroup consisting of alkanols, and are preferably selected from the groupincluding methanol, ethanol, n-propanol, 2-propanol, 1 butanol, and2-butanol.

The resulting product contains a low level of non-modified glycosides,even without the optional steviol glycoside removal step. As usedherein, the expressions “low level non-modified glycosides” or “lowlevel unreacted glycosides” shall refer to glycoside levels of less thanabout 20%, and preferably less than about 15%, on an anhydrous basis. Insome embodiments, an unreacted glycoside level of less than about 12%,less than about 10% or even lower can be attained using this method.Performing the optional step of steviol glycoside removal results ineven lower levels of unreacted steviol glycosides in the final product.

A small part of purified transglucosylated Stevia extract containingα-1,4-glucosyl-derivatives with up to nine α-1,4-glucosyl residues(obtained as described above) was separated and further subjected todecolorizing and desalting treatment (similar to Sample 2) to produceSample 1.

The process used for preparing Sample 2 was repeated without removal ofmaltooligosaccharides from first transglucosylation mixture (containingα-1,4-glucosyl-derivatives with up to nine α-1,4-glucosyl residues).This process yielded Sample 3.

The analysis of each Sample's composition (Table 1) shows a similarcomposition for Samples 1 and 3, whereas Sample 2 had largerconcentration of high glucosyl derivatives (containing up to 20α-1,4-glucosyl residues).

TABLE 1 Composition of glucosyl steviol glycosides samples Content, %Compounds Sample 1 Sample 2 Sample 3 Stevioside 3.1 1.6 2.9 RebaudiosideC 1.0 0.4 1.0 Rebaudioside A 6.1 2.8 5.8 Monoglucosyl-stevioside(StevG1) 7.4 3.7 7.5 Monoglucosyl-Rebaudioside A 11.1 4.5 11.5 (RebAG1)Diglucosyl-stevioside (StevG2) 8.4 4.8 8.8 Diglucosyl-Rebaudioside A 9.65.3 9.7 (RebAG2) Higher Glucosylated derivatives 48.4 57.5 49.3 up to G9Higher Glucosylated derivatives — 15.6 — from G9 to G20 Total content ofunreacted 10.2 4.8 9.7 glycosides Total content of glycosides 95.8 96.296.5

The sensory assessment of samples was carried using aqueous solutions,with 20 panelists. Based on overall acceptance the most desirable andmost undesirable samples were chosen. The results are shown in Table 2.

TABLE 2 Sensory assessment of samples in water system Judgment Sample 1Sample 2 Sample 3 Most desirable  1 18  1 Sweetness power 120 80 120Comments Sweet, slightly bitter, Sweet, light, soft, round, Sweet,slightly bitter, astringent, slight lingering pleasant, similar tosucrose, astringent, slight lingering aftertaste, sweetness onset is nolingering aftertaste, aftertaste, sweetness onset is slow sweetnessonset is rapid slow

As apparent from the results in Table 2, the sweetness quality of theSample 2 was rated as most superior.

The glucosyl stevia composition represented by Sample 2 shows comparablesweetness power (80 times sweeter compared to a 5% sucrose solution)with control Samples 1 and 3 (120 times); however its flavor profile wasclearly superior to the control samples.

The composition can be used as sweetness enhancer, flavor enhancer andsweetener in various food and beverage products. Non-limiting examplesof food and beverage products include carbonated soft drinks, ready todrink beverages, energy drinks, isotonic drinks, low-calorie drinks,zero-calorie drinks, sports drinks, teas, fruit and vegetable juices,juice drinks, dairy drinks, yoghurt drinks, alcohol beverages, powderedbeverages, bakery products, cookies, biscuits, baking mixes, cereals,confectioneries, candies, toffees, chewing gum, dairy products, flavoredmilk, yoghurts, flavored yoghurts, cultured milk, soy sauce and othersoy base products, salad dressings, mayonnaise, vinegar,frozen-desserts, meat products, fish-meat products, bottled and cannedfoods, tabletop sweeteners, fruits and vegetables.

Additionally the composition can be used in drug or pharmaceuticalpreparations and cosmetics, including but not limited to toothpaste,mouthwash, cough syrup, chewable tablets, lozenges, vitaminpreparations, and the like.

The composition can be used “as-is” or in combination with othersweeteners, flavors and food ingredients.

Non-limiting examples of sweeteners include steviol glycosides,stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideD, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside,rubusoside, as well as other steviol glycosides found in Steviarebaudiana Bertoni plant and mixtures thereof, stevia extract, Luo HanGuo extract, mogrosides, high-fructose corn syrup, corn syrup, invertsugar, fructooligosaccharides, inulin, inulooligosaccharides, couplingsugar, maltooligosaccharides, maltodextins, corn syrup solids, glucose,maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugaralcohols.

Non-limiting examples of flavors include lemon, orange, fruity, banana,grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cottoncandy, vanilla flavors.

Non-limiting examples of other food ingredients include flavors,acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilizers, thickeners, gelling agents.

The following examples illustrate various embodiments of the invention.It will be understood that the invention is not limited to thematerials, proportions, conditions and procedures set forth in theexamples, which are only illustrative.

Example 1 Preparation of CGTase

A strain of Bacillus stearothermophilus St-100 was inoculated in 2,000liters of sterilized culture medium containing 1.0% starch, 0.25% cornextract, 0.5% (NH₄)₂SO₄, and 0.2% CaCO₃ (pH 7.0-7.5) at 56° C. for 24hrs with continuous aeration (2,000 L/min) and agitation (150 rpm). Theobtained culture broth was filtered using Kerasep 0.1 μm ceramicmembrane (Novasep, France) to separate the cells. The cell-free permeatewas further concentrated 2-fold on Persep 10 kDa ultrafilters (Orelis,France). The activity of the enzyme was determined according to Hale,Rawlins (1951). A crude enzyme preparation with activity of about 2unit/mL was obtained.

Example 2 Preparation of Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of the reaction mixture was adjusted to pH2.8 by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to the liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzymes.20 grams of activated carbon was added and the mixture was heated to 75°C. and held for 30 minutes. The mixture was filtered and the filtratewas diluted with water to 5% solids content and passed through columnseach packed with 4000 mL Amberlite XAD 7HP macroporous adsorbent resin.The columns were washed with 5 volumes of water and 2 volumes of 20%(v/v) ethanol. The adsorbed glycosides were eluted with 50% ethanol. Theobtained eluate was passed through columns packed with Amberlite FPC23(H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. The ethanol wasevaporated and the desalted and decolorized water solution wasconcentrated at 60° C. under vacuum, then dried into a powder form usinglaboratory spray dryer. 151 grams of product was obtained (Sample 1).

Example 3 Preparation of Glycosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g oftransglucosylated stevia extract obtained according to EXAMPLE 2 wasadded to the liquefied starch and stirred until a homogeneous solutionwas obtained. 200 units of CGTase were added to the solution and themixture was held at a temperature of 65° C. for 24 hours undercontinuous agitation. The obtained reaction mixture was heated at 95° C.for 15 minutes to inactivate the enzyme. 20 grams of activated carbonwas added and the mixture was heated to 75° C. and held during 30 min.The mixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The columns were washedwith 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol. The obtained eluate was passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The ethanol was evaporated and the desaltedand decolorized water solution was concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. 140 grams ofproduct was obtained (Sample 2).

Example 4 Preparation of Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzymes.20 grams of activated carbon was added and the mixture was heated to 75°C. and held for 30 minutes. The mixture was filtered and the filtratewas passed through columns packed with Amberlite FPC23 (H⁺) andAmberlite FPA51 (OH⁻) ion exchange resins. 195 grams of product wasobtained.

Example 5 Preparation of Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g oftransglucosylated stevia extract obtained according to EXAMPLE 4 wasadded to liquefied starch and stirred until a homogeneous solution wasobtained. 200 units of CGTase was added to the solution and the mixturewas held at a temperature of 65° C. for 24 hours under continuousagitation. The obtained reaction mixture was heated at 95° C. for 15minutes to inactivate the enzyme. 20 grams of activated carbon was addedand the mixture was heated to 75° C. and held during 30 min. The mixturewas filtered and the filtrate was diluted with water to 5% solidscontent and passed through columns each packed with 4000 mL AmberliteXAD 7HP macroporous adsorbent resin. The columns were washed with 5volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol. The obtained eluate was passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The ethanol was evaporated and the desaltedand decolorized water solution was concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. 105 grams ofproduct was obtained (Sample 3).

Example 6 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03% for Samples 1 and 3and 0.04% for Sample 2) were blended and dissolved completely in water(up to 100%) and pasteurized. Glucosyl stevia compositions wererepresented by Samples 1, 2, and 3, obtained according to EXAMPLES 2, 3and 5, respectively.

The sensory evaluations of the samples are summarized in Table 3. Thedata show that the best results can be obtained by using the high purityglucosyl stevia composition (containing up to 20 α-1,4-glucosylresidues) (Sample 2). Particularly the drinks prepared with Sample 2exhibited a rounded and complete flavor profile and mouthfeel.

TABLE 3 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel No. 1 Sweet, licorice notes Slight bitterness andNot aftertaste acceptable No. 2 High quality sweetness, Clean, nobitterness and Full pleasant taste similar to no aftertaste sucrose,rounded and balanced flavor No. 3 Sweet, licorice notes Slightbitterness and Not aftertaste acceptable

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

Example 7 Low-Calorie Carbonated Beverage

A carbonated beverage according to formula presented below was prepared.

Quantity, % Ingredients Sample 1 Sample 2 Sample 3 Sucrose 5.5 5.5 5.5Cola flavor 0.340 0.340 0.340 ortho-Phosphoric acid 0.100 0.100 0.100Sodium citrate 0.310 0.310 0.310 Sodium benzoate 0.018 0.018 0.018Citric acid 0.018 0.018 0.018 Rebaudioside A 0.003 0.003 0.003 Glucosylstevia composition 0.05 0.06 0.05 Carbonated water to 100 to 100 to 100

The sensory properties were evaluated by 20 panelists. The results aresummarized in Table 4.

TABLE 4 Evaluation of low-calorie carbonated beverage samples Number ofpanelists detected the attribute Taste attribute Sample No. 1 Sample No.2 Sample No. 3 Bitter taste 10 0 11 Astringent taste 12 0 10 Aftertaste14 0 14 Comments Quality of sweet taste Bitter aftertaste Clean Bitteraftertaste (10 of 20) (20 of 20) (12 of 20) Overall evaluationSatisfactory Satisfactory Satisfactory (3 of 20) (20 of 20) (4 of 20)

The above results show that the beverages prepared using Sample 2possessed the best organoleptic characteristics.

Example 8 Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%)and different glucosyl stevia compositions (0.03% for Samples 1 and 3,and 0.04% for Sample 2) were kneaded well in dough-mixing machine. Theobtained dough was molded and baked in oven at 200° C. for 15 minutes.Glucosyl stevia compositions were represented by Samples 1, 2 and 3,obtained according to EXAMPLES 2, 3 and 5, respectively.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high glucosyl stevia compositioncontaining derivatives with up to 20 α-1,4-glucosyl residues (Sample 2).The panelists noted rounded and complete flavor profile and mouthfeel incookies prepared with Sample 2.

Example 9 Yoghurt

Different glucosyl stevia compositions (0.03% for Samples 1 and 3, and0.04% for Sample 2) and sucrose (4%) were dissolved in low fat milk.Glucosyl stevia compositions were represented by Samples 1, 2 and 3,obtained according to EXAMPLES 2, 3 and 5, respectively. Afterpasteurizing at 82° C. for 20 minutes, the milk was cooled to 37° C. Astarter culture (3%) was added and the mixture was incubated at 37° C.for 6 hours then at 5° C. for 12 hours.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high glucosyl stevia compositioncontaining derivatives with up to 20 α-1,4-glucosyl residues (Sample 2).The panelists noted rounded and complete flavor profile and mouthfeel insamples prepared with Sample 2.

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereof, and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

We claim:
 1. A process for producing a highly purified glucosyl steviacomposition, comprising the steps of: adding starch into water to form astarch suspension; adding a mixture of α-amylase and CGTase into thestarch suspension and incubating for about 0.5 to 2 hours at about75-80° C., resulting in a first liquefied starch suspension;inactivating the α-amylase by low pH heat treatment; cooling the firstliquefied starch suspension and adjusting the pH to about 5.5 to 7.0;adding steviol glycosides into the first liquefied starch suspension,resulting in a first reaction mixture; adding CGTase into the firstreaction mixture and incubating for about 12 to 48 hours at about 55-75°C.; removing non-reactant maltoologosaccharides by contacting the firstreaction mixture with macroporous adsorbent resin and subsequentlyeluting adsorbed diterpene glycosides with aqueous ethanol to result ina glycoside-containing first aqueous ethanol eluate; removing ethanolfrom the first aqueous ethanol eluate, resulting in a first aqueouseluate; concentrating and drying the first aqueous eluate to obtain thefirst dried glucosyl stevia composition; preparing a second liquefiedstarch suspension in the same manner as the first liquefied starchsuspension; adding the first dried glucosyl stevia composition into thesecond liquefied starch suspension, resulting in the second reactionmixture; adding CGTase into the second reaction mixture and incubatingfor about 12 to 48 hours at about 55-75° C.; inactivating the enzyme inthe second reaction mixture by heat treatment; decolorizing the secondreaction mixture; removing non-diterpene compounds by contacting thedecolorized second reaction mixture with macroporous adsorbent resin andsubsequently eluting adsorbed diterpene glycosides with aqueous ethanolto result in a glycoside-containing second aqueous ethanol eluate;desalting the glycoside-containing second aqueous ethanol eluate withion-exchange resins; removing ethanol from the second aqueous ethanoleluate, resulting in a second aqueous eluate; and concentrating anddrying the second aqueous eluate to obtain the highly purified glucosylstevia composition; wherein the highly purified glucosyl steviacomposition comprises steviol glycosides and their derivatives having upto twenty α-1,4-glucosyl residues.
 2. The process according to claim 1,wherein the mixture of α-amylase and CGTase contains about 0.001-0.2 KNUof α-amylase per one unit of CGTase.
 3. The process of claim 2, whereinthe mixture of α-amylase and CGTase contains about 0.05-0.1 KNU ofα-amylase per one unit of CGTase.
 4. The process according to claim 1,wherein the weight of added steviol glycosides is about equal to that ofthe starch used to make the first liquefied starch suspension.
 5. Theprocess according to claim 1, wherein the added steviol glycosides areselected from the group consisting of stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, as well asother steviol glycosides found in Stevia rebaudiana Bertoni plant andmixtures thereof.
 6. The process according to claim 1, wherein theweight of added first dried glucosyl stevia composition is about equalto that of the starch used to make the second liquefied starchsuspension.
 7. The process according to claim 1, wherein the first driedglucosyl stevia composition comprises stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, andα-1,4-glucosyl derivatives thereof with up to nine α-1,4-glucosylresidues.
 8. The process according to claim 1, wherein the CGTase isproduced by cultures of Bacillus stearothemophilus.
 9. The processaccording to claim 1, wherein the CGTase is added at amount of about0.2-4 units per gram of solids.
 10. The process according to claim 1,wherein the CGTase is added at amount of about 0.5-1.2 units per gram ofsolids.
 11. The process according to claim 1, wherein the decolorizingis performed using activated carbon.
 12. The process according to claim1, wherein the decolorizing is performed using ion exchange resins ormembranes, said membranes being selected from the group consisting ofultrafiltration, nanofiltration, and reverse osmosis membranes.
 13. Theprocess of claim 1, wherein removing maltooligosaccharides andnon-diterpene compounds is conducted with a plurality of sequentiallyconnected columns packed with a macroporous adsorbent resin, followed bywashing the columns with water, then washing with about 10-50% (v/v)ethanol, disconnecting the columns, and then eluting each columnindividually with 30-100% ethanol.
 14. The process according to claim 1,wherein the desalting is performed by passing the eluate through columnspacked with ion exchange resins or membranes, said membranes beingselected from the group consisting of ultrafiltration, nanofiltration,and reverse osmosis membranes.
 15. The process according to claim 1,wherein the highly purified glucosyl stevia composition has at leastabout 95% total steviol glycosides on an anhydrous basis.
 16. Theprocess according to claim 1, wherein the highly purified glucosylstevia composition has less than 10% unreacted steviol glycosides on ananhydrous basis.
 17. A sweetener composition comprising a highlypurified glucosyl stevia composition made by the process of claim 1, andan additional sweetening agent selected from the group consisting of:stevia extract, steviol glycosides, stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, other steviolglycosides found in Stevia rebaudiana Bertoni plant and mixturesthereof, Luo Han Guo extract, mogrosides, high-fructose corn syrup, cornsyrup, invert sugar, fructooligosaccharides, inulin,inulooligosaccharides, coupling sugar, maltooligosaccharides,maltodextins, corn syrup solids, glucose, maltose, sucrose, lactose,aspartame, saccharin, sucralose, sugar alcohols, and a combinationthereof.
 17. A flavor composition comprising a highly purified glucosylstevia composition made by the process of claim 1, and an additionalflavoring agent selected from the group consisting of: lemon, orange,fruit, banana, grape, pear, pineapple, mango, bitter almond, cola,cinnamon, sugar, cotton candy, vanilla, and a combination thereof.
 19. Afood ingredient comprising a highly purified glucosyl stevia compositionmade by the process of claim 1, and an additional food ingredientselected from the group consisting of: acidulants, organic and aminoacids, coloring agents, bulking agents, modified starches, gums,texturizers, preservatives, antioxidants, emulsifiers, stabilizers,thickeners, gelling agents, and a combination thereof.
 20. A food,beverage, cosmetic or pharmaceutical product comprising a highlypurified glucosyl stevia composition made by the process of claim 1.